Our long term objectives are to study the development and regeneration of skin ectodermal organs and to apply the principles toward regenerative medicine. Over the last two decades, my laboratory has made a long term commitment to this goal; we have produced several basic scientific discoveries and made conceptual progress toward the advancement of skin regenerative biology. In the next phase, we will consolidate new findings from two recently completed RO1 grants to focus on deciphering the mechanism of phenotypic specification in skin organogenesis, a major challenge in the next phase of cutaneous regenerative medicine. Through progress in stem cell biology and reprogramming technology, scientists are now able to generate epidermal progenitors, in the form of dissociated cells or a keratinocyte monolayer. How to guide these progenitors to form skin with different architecture remains unknown. Understanding these processes is crucial to ensure proper regeneration of a wound graft. A fundamental feature of the skin is the striking regional variation, where distinct skin types (e.g., facial skin and scalp glabrous skin, etc) develop at different body regions to serve different purposes.2,3 We postulate that competent, dissociated, epidermal progenitors interact with dermal cells to form reconstituted skin, a process which is regulated by environmental signals which change over developmental time to generate specific skin appendage phenotypes. We will focus on three fundamental aspects, using the most appropriate animal models for each process. Mouse and human cells are used in Aim 1 whereas chicken skin is used for Aims 2 and 3 because it features remarkable regional differences ideal for experimental analyses. In Aim 1, we will take a multi-disciplinary approach to study how the reconstituted skin layer is generated from dissociated cells. Time-lapse imaging, transcriptome analyses and molecular perturbation will be used to study how the dissociated cells can self-assemble into a layered configuration via a series of multicellular morphological transitions. We will focus on the roles of MMPs in this morphogenetic transition and will attempt to reactivate the morphogenetic ability in adult mouse and human cells by modifying the Wnt-MMP-ECM module. In Aim 2, we will study how skin progenitors are specified during regionalization. Our preliminary data suggests that the conversion of scales to feathers proceeds in a stepwise manner via a hierarchy of signaling molecules. Based on preliminary studies, we will focus on the role of Sox genes in establishing this hierarchy. In Aim 3, we will study the morphogenetic principles of organ shaping. We will focus on the regulation of appendage length using feathers as a model. We will evaluate the hypothesis that complex organ shapes are specified by a core circuit that defines a prototypic phenotype qualitatively and modulator circuits that modify specific dimensional parameters quantitatively in a temporal-spatial manner. We hypothesize that FGF and Zic signaling, respectively, may be examples of such molecular circuits.